Methodology

The copy number of methylated O6-Methylguanine-DNA Methyltransferase (MGMT) is determined relative to the copy number of beta-actin (ACTB) by real-time methylation specific polymerase chain reaction (MSP, MDxHealth, assay version 2). A total of 40 µm FFPE glioma tissue is used for DNA extraction. Prior to DNA extraction, tumor tissue is enriched by means of manual macrodissection. A maximum of 1500 ng DNA is treated with Sodium Bisulfite which causes unmethylated Cytosines to be converted to Uracyl while methylated Cytosines remain unchanged. Subsequently, MSP is performed on the ABI7900HT instrument to quantify the copy number of MGMT/ACTB (1).

Clinical Implication

MGMT is a DNA repair enzyme that is frequently deactivated by hypermethylation in human cancer. When cells lack MGMT activity, DNA damage is not repaired properly making an individual more prone to the development of cancer. Of interest, this lack of MGMT activity also makes a tumor cell more sensitive to radiation therapy and certain alkylating drugs such as temozolomide (2). In the international prognostic Phase III (RTOG 0525) validation study, MDxHealth’s MGMT test successfully identified newly diagnosed glioblastoma patients who are more likely to live longer and have a longer progression free time period following treatment with temozolomide (3).

Specimen Requirements

Acceptable specimens for the assay are formalin-fixed, paraffin-embedded glioma tissue specimens with a fixation time of 6-48 hours.

Storage and Shipment Instructions

Maintain and ship specimens at ambient temperature.

Limitations

Some samples do not show significant amplification of ACTB. This is due either to the sample being very small, or its DNA being degraded. There are specifications for the number of detected copies of ACTB, and if these are not met the sample is deemed invalid. Fixatives other than formalin or prolonged fixation time may give rise to inadequate results.